Predicting Protein Evolution with Phage Escape

通过噬菌体逃逸预测蛋白质进化

• 批准号: 7860354
• 负责人: TOBIN J DICKERSON
• 金额: $ 23.74万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7860354
• 关键词: Affect; Amino Acid Sequence; Antibodies; Antigens; Anxiety; Area; Avian Influenza; Bacteriophage M13; Bacteriophages; Binding; Biological; Biological Models; Cell model; Cells; Collection; Cytotoxic T-Lymphocytes; Diversity Library; Drug resistance; Ensure; Epitopes; Escape Mutant; Evolution; Experimental Designs; Future; Generations; Genes; Genomic Segment; Goals; H5 hemagglutinin; Hemagglutinin; Human; Immune; Immune response; Immune system; Immunity; In Vitro; Infection; Influenza; Invaded; Laboratories; Libraries; Ligand Binding; Light; Mammalian Cell; Membrane Glycoproteins; Membrane Proteins; Methods; Mind; Molecular; Molecular Biology Techniques; Monoclonal Antibodies; Mutate; Mutation; Peptide Sequence Determination; Phage Display; Play; Procedures; Process; Protein Region; Proteins; Public Health; Research; Scanning; Scheme; Screening procedure; Side; Solutions; Staging; System; Techniques; Technology; Time; Variant; Viral; Virulent; Virus; base; combat; combinatorial; experience; improved; in vivo; influenza outbreak; influenzavirus; member; mortality; mutant; neutralizing antibody; pandemic disease; pandemic influenza; pathogen; pressure; prevent; protein function; public health relevance; research study; response; sialic acid receptor

DESCRIPTION (provided by applicant): In light of the extreme mortality rates associated with human infection with avian influenza strains, the emergence of a pandemic influenza outbreak is of immense concern. Pandemic strains are thought to occur through the introduction of genetically divergent protein sequences by reassortment of viral genomic segments during coinfection with multiple influenza viruses (antigenic shift), as well as gradual accumulation of mutations sufficient to alter the antigenicity of a given strain such that preexisting host immunity becomes ineffective (antigenic drift). The goal of this project is to mirror these processes in vitro by using an unbiased combinatorial library approach, thereby allowing access to large sections of antigenic space. Specifically, we will utilize phage escape technologies developed in our laboratory to generate combinatorial libraries of the viral surface glycoprotein hemagglutinin (HA) to allow access to the sequence space available to the influenza virus. The essence of our tactic is an alternating scheme of screening for human antibodies which first prevent HA binding to cells, followed by a selection for mutant HA proteins that "escape" our antibodies and remain bound to cells. Repeated iteration of this process will reveal an array of varying HA clones capable of binding to cells, as well as the set of neutralizing antibodies corresponding to each HA. Ultimately, we envision that our approach will allow for an assessment of both the current avian influenza strain (H5), virulent HA mutants which occurred in the past (e.g., H1), as well as those that have not yet emerged. Furthermore, inherent to our experimental design is a concurrent identification of specific human antibodies that neutralize HA binding to host cells. PUBLIC HEALTH RELEVANCE: Rarely does a year go by that influenza is not a public health concern, and in particular, the threat of a pandemic avian influenza in recent years has caused immense anxiety in the minds of the public. Our proposal utilizes modern molecular biology techniques to mimic the evolution of the influenza protein required for infection. We aim to generate a laboratory system that can predict influenza strains and in the process, develop neutralizing therapies for these strains before they can become a threat to public health.

描述（由申请人提供）：鉴于与人类感染禽流感菌株相关的极端死亡率，大流行性流感爆发的出现引起了极大的关注。人们认为，通过与多种流感病毒（抗原转移）共同感染病毒基因组片段的重新分配，通过重新分配病毒基因组片段（抗原转移）以及足以改变给定应变的抗原性抗原性，使宿主免疫的抗原性变得蚂蚁（Antigection n eftercection）（抗原）（蚂蚁）抗原性（抗原）（抗原性）（抗原性）（抗原性），这是通过重新感染的，可以通过多种流感病毒（抗原转移）来逐渐膨胀（抗原性）（抗原性），这是通过在遗传上发散蛋白序列引入遗传学分歧的蛋白质序列发生的。该项目的目的是通过使用公正的组合库方法在体外反映这些过程，从而允许访问大型抗原空间。具体而言，我们将利用在实验室中开发的噬菌体逃生技术来生成病毒表面糖蛋白糖蛋白hemagglutinin（ha）的组合文库，以允许进入流感病毒可用的序列空间。我们策略的本质是对人类抗体进行筛选的交替方案，该抗体首先防止HA与细胞结合，然后选择“逃脱”我们的抗体并保持与细胞结合的突变蛋白选择。该过程的重复迭代将揭示能够与细胞结合的一系列不同的HA克隆以及与每个HA相对应的中和抗体的集合。最终，我们设想我们的方法将允许评估过去发生的当前禽流感（H5），有毒的HA突变体（例如H1）以及尚未出现的突变体。此外，我们的实验设计固有的是对特定人类抗体的同时识别，该抗体中和HA与宿主细胞的结合。公共卫生相关性：流感很少一年并不是一个公共卫生的关注，尤其是近年来大流行禽流感的威胁引起了公众的极大焦虑。我们的建议利用现代分子生物学技术模仿感染所需的流感蛋白的演变。我们旨在产生一个可以预测流感菌株的实验室系统，在此过程中，在这些菌株成为对公共卫生的威胁之前，为这些菌株开发中和疗法。